Multicentre genetic diversity study of carbapenem-resistant Enterobacterales: predominance of untypeable pUVA-like blaKPC bearing plasmids

Abstract Objectives Carbapenem-resistant Enterobacterales (CRE) are an urgent public health threat. A better understanding of the molecular epidemiology and transmission dynamics of CRE is necessary to limit their dissemination within healthcare settings. We sought to investigate the mechanisms of resistance and spread of CRE within multiple hospitals in Maryland. Methods From 2016 to 2018, all CRE were collected from any specimen source from The Johns Hopkins Medical Institutions. The isolates were further characterized using both phenotypic and genotypic approaches, including short- and/or long-read WGS. Results From 2016 to 2018, 302 of 40 908 (0.7%) unique Enterobacterales isolates were identified as CRE. Of CRE, 142 (47%) were carbapenemase-producing CRE with KPC (80.3%) predominating among various genera. Significant genetic diversity was identified among all CRE with high-risk clones serving as major drivers of clonal clusters. Further, we found the predominance of pUVA-like plasmids, with a subset harbouring resistance genes to environmental cleaning agents, involved in intergenus dissemination of blaKPC genes. Conclusions Our findings provide valuable data to understand the transmission dynamics of all CRE within the greater Maryland region. These data can help guide targeted interventions to limit CRE transmission in healthcare facilities.


Introduction
The CDC Antibiotic Resistance Threats in the United States, 2019 report highlights that antimicrobial resistance contributes to over 2.8 million infections and greater than 35 000 deaths annually in the USA. 1 Of antimicrobial-resistant organisms, the CDC has assigned the highest threat level to carbapenem-resistant Enterobacterales (CRE), declaring they require urgent public health attention. 1,2 A better understanding of the molecular epidemiology and transmission dynamics of CRE is necessary to limit their dissemination within healthcare settings.
There are two major mechanisms driving the spread of CRE in the USA: (i) vertical transmission of successful clones [e.g. ST258 Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae] and (ii) horizontal spread of carbapenemase-producing CRE (CP-CRE) through promiscuous plasmids and/or associated mobile genetic elements (e.g. IncFII, pKpQIL, Tn4401). 3 To date, a primary focus of public health efforts has been on CP-CRE because their carbapenemase genes can spread with great ease across various Gram-negative species or even between genera through transmissible genetic elements. 4 However, we believe both mechanisms merit further investigation to understand their relative importance in contributing to the CRE epidemic. Moreover, studies of CRE transmission in the USA have generally been limited to outbreak settings or have focused on specific species or carbapenemase genes resulting in incomplete data for a comprehensive understanding of CRE transmission dynamics. 5,6 Our objective was to understand the mechanisms of resistance and spread that contribute to the successful dissemination of CRE, regardless of species, encountered within hospital settings. These data are critical in guiding interventions to limit CRE transmission in acute care facilities. . Carbapenem antimicrobial susceptibility testing (AST) results were determined clinically using the BD Phoenix Automated System (BD Diagnostics, Sparks, MD, USA) and interpreted following CLSI guidelines. 7 The phenotypic modified carbapenem inactivation method (mCIM) was performed on all CRE isolates to differentiate CP-CRE from non-carbapenemaseproducing CRE (non-CP-CRE). 7 All CRE isolates were stored frozen using Microbank beads (Pro-Lab Diagnostics, Richmond Hill, ON, Canada) at −80°C until further testing was pursued. This study was approved by The Johns Hopkins University School of Medicine institutional review board.

AST
Unique patient isolates (i.e. one isolate per patient per species) were subcultured from frozen stock along with a second subculture on 5% sheep blood agar prior to performing AST. Repeat AST was performed on isolates using lyophilized Sensititre broth microdilution (BMD) CTNX2F or GN7F and MDRGNX2F panels (Thermo Fisher Scientific, Waltham, MA, USA). Results were interpreted following CLSI guidelines with the exception of eravacycline, omadacycline, plazomicin and tigecycline, which were interpreted applying FDA susceptibility interpretive criteria. 8 Quality control (QC) was set up on each day of testing.

Plasmid assemblies
pUVA-like plasmids were assembled using Canu v 1.7. Draft assemblies were polished with Illumina short reads in response to iterative Pilon runs. 9,10 Plasmids were circularized and rotated to match the origin of replication of the reference pUVA01 plasmid. 17,18 Plasmids were annotated using Prokka. 13 Multiple sequence alignments were generated using MAFFT, and phylogenetic trees were constructed using phyML to compute the sequence similarity of plasmids harbouring bla KPC . 19,20 Additionally, a whole plasmid phylogenetic tree was constructed to visualize the sequence similarity of the plasmids. To identify structural variations in plasmid sequences with respect to the reference pUVA01 plasmid, full-length plasmid sequences were aligned to the pUVA01 reference using the nucmer utility (MUMmer3 package). 21 Structural variants in the plasmid assemblies were detected and corrected (Assemblytics software). 22 Variations found using nucmer and assemblytics were plotted using Circa (http://omgenomics.com/circa/), relative to the pUVA01 reference sequence.

Comparison of pUVA-like plasmids
For comparison, 42 092 RefSeq plasmid sequences were downloaded and aligned to the reference pUVA01 plasmid using minimap2 and followed by Bowtie2. 10,19 A phylogenetic tree was generated based on sequence similarity using Mash for the subset of RefSeq plasmids that aligned to pUVA01. 23 The resulting RefSeq plasmids matching pUVA01 were compared against the ∼10 kb insertion from the KLPN_262 assembled plasmid using the nucmer utility to identify any plasmids with similar structural variants. 17

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Genetic diversity of CRE

Phylogeny among CRE
Focusing on the top three CRE encountered, significant heterogeneity was identified among bacterial STs (Figure 1) (13)]. Outside of ST258, a few additional clusters of STs were identified across K. pneumoniae complex isolates. Three (2.5%) CR-K. pneumoniae complex isolatesall of which were non-CP-CRE-belonged to ST37. Additionally, six (5.0%) KPC-3-producing K. pneumoniae complex belonged to ST340. A small cluster (n = 5) of KPC-2-producing ST15 K. pneumoniae complex isolates was identified in 2016 and persisted over the next 2 years. Overall, STs were diverse, with 31 different STs identified among non-CP and 22 STs among CP-K. pneumoniae complex (Figure 1a). SNP analysis revealed between 0 and <1000 SNPs identified among the core genome of K. pneumoniae complex isolates. Significant ST diversity was identified among CR-E. cloacae complex isolates, with 29 STs among non-CP and 13 STs among CP isolates (Figure 1b). Two small clusters of three isolates and two isolates of CP-E. cloacae complex ST93 and ST316, respectively, were identified. The predominant ST amongst non-CP-CRE E. cloacae complex isolates was ST108 (seven, 11.5%), with small clusters of two isolates each of ST32, ST66, ST113, ST533 and ST807. Isolates differed between 0 and <139 000 SNPs among the core genome of E. cloacae complex isolates.

Untypeable RepA plasmid analysis
The predominance of bla KPC carriage by the untypeable RepA plasmid led to further investigations of this plasmid. The untypeable RepA plasmid, present in 43.9% of KPC producers, shared a similar backbone to pUVA01, plasmids that caused an intergenus CRE outbreak of KPC at a facility in Virginia, but still demonstrated variability with multiple recombination, insertion and deletion events. 18 In our cohort, the untypeable RepA plasmids clustered into two groups based on phylogenetic similarity (Figure 2).
The first group of 12 untypeable RepA plasmids varied from 40 to 83 kb in size and lacked the Tn4401 element and were distantly related to the pUVA02 plasmid (Figure 2, KLPN_647 representative plasmid). They harboured bla KPC-3 and were isolated from nine K. pneumoniae complex of various STs (ST11, ST15, ST16, ST273, ST340), two E. coli and a single E. cloacae complex. A number of single base insertions and deletions occurred throughout the untypeable RepA plasmids, compared with pUVA01. These mutations generally occurred in the region of plasmid replication and maintenance genes. For all plasmids in this cluster, a deletion was observed in the virB4 gene, which encodes an inner membrane ATPase that is part of the type IV secretion system responsible for conjugative transfer.
The second, larger cluster of untypeable RepA plasmids closely resembled the pUVA01 plasmid and included 34 plasmids ranging in size from 40 to 57 kb (Figure 2; KLPN_262 representative plasmid). These plasmids harboured bla KPC-3 (24, 71%) or bla KPC-2 (10, 29%). Organisms within this cluster of plasmids were identified in more diverse species than the first cluster and included K. pneumoniae complex (17) KLPN_647: Cluster 1 representative untypeable RepA plasmid isolated from a Klebsiella pneumoniae isolate. Note the deletion observed in the virB4 gene, which was present among all plasmids in this cluster. KLPN_262: Cluster 2 representative untypeable RepA plasmid isolated from a K. pneumoniae complex isolate. Note the 10 kb insertion containing the bin3 gene and frmRAB operon. Outer track is the gene identification with gene boundaries highlighted in multiple colours, blue line is the Tn4401 element found in Cluster 2 plasmids (at 28-51k), the black track highlights the read coverage at the position, and the inner track highlights structural variations compared with pUVA01. Structural variants are colour coded as red for insertion, green for deletion and pink for repeat expansion. The green arcs are the deletion track and the red arcs are the insertion track. complex (2), Pantoea species (1), K. oxytoca (1) and S. marcescens (1). Interestingly, 13 of the 34 plasmids (38%) acquired a 7-10 kb insertion that contained bin3 (recombinase), frmA, frmB and/or frmR genes. frm genes are associated with aldehyde resistance and likely contribute to plasmid survival in the hospital environment where disinfectants containing aldehydes or chlorine are commonly used.
Of 42 092 RefSeq plasmids, 5001 plasmids successfully aligned against the reference pUVA01 plasmid using minimap2. The more sensitive alignment using Bowtie2 yielded only 104 RefSeq plasmids matching the reference pUVA01. When compared with the assembled representative KLPN_262 plasmid using nucmer, 14 of these RefSeq sequences contained the same ∼10 kb insertion. Figure 3 shows the phylogenetic tree for the 104 RefSeq plasmids matching the pUVA01 reference plasmid, alongside pUVA01, pUVA02, KLPN_262 and KLPN_647.

Discussion
Our findings demonstrate high genetic diversity among CRE and further define the successful establishment of a promiscuous bla KPC -bearing plasmid among various Enterobacterales within the hospital setting. Interestingly, a subset of the bla KPC -bearing pUVA-like plasmids demonstrated a 10 kb insert encoding resistance genes to environmental cleaning agents. These data can inform effective approaches to limit CRE transmission in healthcare settings, including infection prevention and antibiotic stewardship strategies.
Overall, the proportion of unique Enterobacterales identified in our cohort was stable and remained at <1% throughout the 3 year study period, consistent with data from the CDC indicating stabilization of CRE rates from 2013 to 2017. 1 The plateauing of CRE prevalence is likely a reflection of successful, targeted Figure 3. Phylogenetic tree demonstrating the genome distances as calculated by Mash 23 between the 104 RefSeq plasmids that align to the reference pUVA01 plasmid, the representative assembled KLPN_262 and KLPN_647 (purple), and the reference pUVA01 and pUVA02 (blue). Highlighted in green is the subset of 14 plasmids that contain the same ∼10 kb insertion identified in the KLPN_262 cluster of assembled plasmids. infection prevention efforts implemented at both local and national levels to curb their spread. As an example, the CDC has advocated screening for CP-CRE colonization upon hospital admission for patients who had overnight stays in healthcare facilities outside the USA in the prior 6 months. 4 Similarly, they have recommended communicating to other facilities a patient's CP-CRE status during transitions of care to limit their spread across the healthcare continuum.
K. pneumoniae complex has been implicated as the Enterobacterales species with the greatest contribution to the CRE epidemic. Interestingly, we found a significant decrease in the proportion of K. pneumoniae complex isolates identified as CR over the study period. Our data are in agreement with those of Wilson et al., 24 who reported decreasing proportions of CR-K. pneumoniae complex across US Veterans Health Administration facilities from 2006 to 2015. They also reported an increase or a 'second epidemic' wave of CR-E. cloacae complex isolates originating from the West Coast and progressing eastward across the USA. The proportion of CR-E. cloacae complex was stable in our cohort, but it is yet unclear if this so-called 'second epidemic' will impact the mid-Atlantic region.
We observed an even split of carbapenem resistance mediated by CP and non-CP mechanisms across our cohort. This is similar to previous reports from our group from 2013 to 2016. 25,26 This distribution varies by organism, with K. oxytoca, C. freundii complex and K. pneumoniae complex most commonly identified as CP-CRE, whereas P. mirabilis, M. morganii, K. aerogenes and E. cloacae complex were more likely to be non-CP-CRE. Clinical microbiology laboratories are encouraged to generate similar regional data to inform prioritization of carbapenemase detection in their facilities. Further, implementation of modalities with rapid turnaround times (e.g. same-day detection of carbapenemase production by phenotypic or molecular methods) should be considered for Enterobacterales most likely to be CP-CRE, to limit local dissemination and aid in patient management.
High genetic diversity was observed among all genera of CRE indicating independent mutational events through antibiotic selection pressure, clonal spread and mobile genetic elements all playing a role in the dissemination and molecular evolutionary trajectory of CRE. We observed the establishment of successful clones among our CRE including ST131 E. coli and ST258 K. pneumoniae complex among both non-CP-CRE and KPC-producing isolates. In contrast to a recent report highlighting the emergence of the K. pneumoniae complex ST307 clone in Texas, we identified only a single isolate belonging to this ST throughout the period of our cohort. 27 The introduction of KPC-3 among ST131 E. coli raises concerns because of its international success and the global dominance of CTX-M-15 among ST131, resulting in considerable morbidity and mortality. 28 Interestingly, ST108 E. cloacae complex, a clone previously associated with ESBL production, predominated in our acute care facilities. 29 The ST108 cluster in our facilities were non-CP-CRE and did not produce ESBLs. Another US report found that ST171 was the predominant CR E. cloacae complex clone, underscoring important institutional differences in established clones conferring carbapenem resistance. 5 In addition to clonal spread of successful CP-CRE clones, independent acquisition events of bla KPC were notable and linked to an untypeable RepA plasmid bearing bla KPC . 30 Our findings differ from reports from the UK noting the importance of an IncFII pKpQIL-like plasmid as well as IncR, ColRNAI and IncX3 plasmids contributing to the spread of bla KPC . Although we identified similar associations with IncF and IncR plasmids and bla KPC , the untypeable RepA plasmid predominated. The untypeable RepA plasmid identified in our cohort is closely related to the pKPC-UVA01/02 plasmids reported to cause an outbreak of KPC among isolates in four genera of Enterobacterales in Virginia, a US state adjacent to Maryland. 17,18 Similarly, we identified two separate clusters of diverse pUVA-like plasmids among nine different genera associated with and without the transposition element Tn4401. Unlike previous reports, we identified the pKPC-UVA01-like plasmids among successful high-risk clones (ST258 and ST131). 18 The combination of a broad host range and highly mobile plasmids among dominant clones is concerning. Furthermore, we demonstrated the evolution of the pUVA-like plasmids, even within a defined 1 month outbreak that we previously reported on, with mutations occurring in conjugative transfer genes. 31 An interesting finding in our cohort was the detection of a 7-10 kb segment of the pUVA-01-like plasmids that harbours a recombinase gene (bin3) and frmRAB operon. The frm genes encode enzymes that function to detoxify a variety of compounds, such as S-formylglutathione hydrolase (frmB) and S-(hydroxymethyl) glutathione dehydrogenase ( frmA), which are involved in resistance to and detoxification of formaldehyde and chlorine. 32-34 The acquisition of these genes may contribute to the survival of isolates harbouring pUVA-01-like plasmids in the hospital environment where disinfectants containing aldehydes or chlorine are frequently used. Additionally, the use of these agents may also select for further horizontal transmission of bla KPC among diverse genera. CRE survive in the hospital environment despite stringent environmental cleaning and disinfection. 31,35 This is concerning because there are persistent reservoirs in the hospital setting for these pathogens to thrive, including sink drains in bathrooms, where environmental cleaning may lead to further selection, evolution and persistencefurthering intergenus spread and success in the hospital environment. 36 Novel methods for cleaning and irradiation of organisms harbouring successful plasmids should be a focus of future interventions.
Our study has limitations. First, inclusion of isolates was limited to the greater Maryland region of the USA and may not be generalizable to other regions. However, our study is unique in including diverse hospitals in terms of bed size and patient population whereas current CRE epidemiology studies generally focus on large academic centres. Second, application of a hybrid approach including short-and long-read genome assemblies among CP-CRE isolates to study plasmid dynamics limits our understanding of the evolution of plasmids to those harbouring carbapenemase genes. Our findings underscore the importance of understanding the evolution of plasmids with and without resistance genes. Future studies should focus on analysing plasmids in all CRE. Similarly, a recent study highlighted the modular exchange that occurs among plasmids that harbour resistance genes and those that do not. 30 Third, the acquisition of aldehyde and chlorine resistance genes identified among the untypeable RepA plasmids requires further investigation to understand the contribution to environmental persistence. Last, despite passing QC, an alert was sent out to customers of the Sensititer MDRGNX2F panels that the panels utilized in this study may produce lower cefiderocol MICs for some Gram-negative organisms.
Our findings provide valuable data to understand the transmission dynamics of all CRE within the greater Maryland region. Further, we demonstrate the predominance of bla KPC pUVA-like plasmids with a subset harbouring resistance to environmental cleaning agents, potentially contributing to their persistence and dissemination in the healthcare setting. These data are significant to guide infection prevention efforts to reduce the development and sustainability of CRE within healthcare facilities.

Funding
This work was supported by an National Institutes of Health R21-AI130608 awarded to P.J.S.